Feline bronchioloalveolar lung carcinoma xenograft and cell line

ABSTRACT

The present invention provides feline transplantable lung carcinoma xenografts and cell lines. Such xenografts and cell lines exhibit a number of unique characteristics which allows their use in experimental models of carcinoma in order to dissect out the molecular basis of this phenotype. This experimental model of carcinoma can be used to identify molecular targets for therapeutic intervention and to assess the efficacy of a broad spectrum of diagnostic and therapeutic agents. Specific animal models of lung cancer are described as well as methods for evaluating diagnostic and therapeutic agents for treating lung cancer.

RELATED APPLICATIONS

This application claims priority under Section 119(e) from U.S.Provisional Application Ser. No. 60/293,643 filed May 25, 2001, thecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the use of feline lung carcinomaxenografts and cell lines as models for both the evaluation of thecancer phenotype as well as the generation of novel diagnostic andtherapeutic methods for the clinical management of this pathology.

BACKGROUND OF THE INVENTION

Over the past decade, there has been a marked increase in both thenumber of new cases and in deaths related to lung cancer nationwide.Tobacco (cigarette smoking) has been implicated in the initiation andpromotion of the majority of these new cases of lung cancer. Carcinogensand tumor promoters in cigarette smoke are thought to be responsible formany of the genetic and epigenetic alterations in bronchial and alveolarepithelial cells that result in the multistep progression into lungcancer.

Peripheral adenocarcinoma (PAC) and bronchioloalveolar carcinoma (BAC)are forms of lung cancer whose etiology and pathogenesis arecontroversial and whose link to either main stream tobacco smoking orsecond hand smoking unproven. While squamous cell carcinomas and smallcell carcinomas have shown an overall decrease in incidence in the pastdecade, peripheral adenocarcinomas (PACs) and bronchioloalveolar lungcancers (BACs) have shown an exponential increase (Barsky et al., Cancer1994; 73: 1163–1170; Barsky et al., Modern Path 1994; 7: 633–640). Theseincreases have been observed equally in both smokers as well asnon-smokers. These epidemiological observations provide evidence thateither different etiological factors exist (other than main stream orsecond hand smoke) that cause PACs and BACs or that differentetiological co-factors that are synergistic with main stream or secondhand smoke play a role in the genesis of PAC/BAC. Some of thedistinguishing pathological, biological epidemiological and perhapsetiological features which distinguish PAC/BAC from other types of lungcancer are its peripheral location, its association with desmoplasia(scarring) (Barsky et al., Am J Pathol 1986; 124:412–419), itssignificant occurrence in non-smokers, its comparatively highfemale/male ratio, and its tendency to appear in multiple and bilateralfoci (especially BAC). Although some theories suggest that thismultifocality is due to intrapulmonary metastatic spread or acombination of aerosolization and aspiration, we have demonstrated thatat least a partial basis for this multifocality or multicentricity ismulticlonality (Barsky et al., Cancer 1994; 73: 1163–1170; Barsky etal., Modern Path 1994; 7: 633–640).

The multifocal nature of BAC was first described in 1876 Malassez etal., Archives of Physiology and Normal Pathology 1876; 3:353–372). BACwas further defined as a well-differentiated adenocarcinoma occurring inthe periphery of the lung which tended to spread along aerogenous andlymphatic routes within the confines of the lung (Liebow,Bronchiolar-alveolar carcinoma. Adv Intern Med 1960; 10:329–358). In therevised WHO lung tumor classification (ICD-0) (World HealthOrganization. Histological typing of lung tumors. Internationalhistological classification of tumors, 2nd ed. Geneva. WHO. 1981),bronchioloalveolar carcinoma was included as a subtype ofadenocarcinoma. Although there has been considerable debate as towhether bronchioloalveolar carcinoma represents a distinct clinicalentity separate from other adenocarcinomas (Schraufnagel et al., Am RevResp Dis 1982; 125:74–79), recent evidence suggests differences inincidence trends, survival, and sex and age distributions compared toother adenocarcinomas and other lung cancer cell types (Grover et al.,Ann Surg 1989; 209:779–90). For these reasons BAC should be consideredas a distinct clinicopathological entity. Pathologists categorize BACinto mucinous and non-mucinous on the basis of their microscopicappearance and further subcategorize non-mucinous BAC into Clara, TypeII pneumocyte and mixed cell of origin on the basis of ultrastructuraland immunocytochemical findings (Yoneda K Cancer 1990;164–169).

Peripheral adenocarcinoma has long been the predominant cell type amongfemale lung cancers (Wynder et al., Eur J Clin Oncol 1987; 23:1491–1496)and there is abundant evidence that this cell type has been increasingin both men and women (Vincent et al., Cancer 1977; 39:1647–1655; Doddset al., JNCI 1986; 76:21–29; Valaitis et al., Cancer 1981;47:1042–1046;Percy et al., Lung cancer: causes and prevention. Mizell M, Correa P,eds. Verlag Chemie International Inc. 1984). According to SEER data for1973–1981 (Percy et al., Lung cancer: causes and prevention. Mizell M,Correa P, eds. Verlag Chemie International Inc. 1984), the incidence ofadenocarcinoma has increased 3% per year in males compared to 1.50% peryear in females. In 1981, 25% of white male lung cancers and 35% ofwhite female lung cancers were adenocarcinoma. Increases in theproportion of adenocarcinomas have also been reported in Japan (Tsuganeet al., Jpn J Can Res 1987; 78:162–169; Watanabe et al., Jpn J CancerRes 1987; 78:460–6) and Israel (Rennert et al., Cancer Det Prev 1991;15:99–101). Few studies report the proportion of bronchioloalveolarcarcinomas separately from other adenocarcinomas. In SEER data (Percy etal., Lung cancer: causes and prevention. Mizell M, Correa P, eds. VerlagChemie International Inc. 1984), 17% of adenocarcinomas werebronchioloalveolar carcinomas, 15% were other subtypes, and 68% wereclassified adenocarcinoma NOS. While bronchioloalveolar carcinoma (BAC)represented approximately 3.5% of all lung cancers in the SEER dataPercy et al., JNCI 1983; 70:663–666), other studies have reported thisproportion to vary from 1% to 8% (Liebow “. Bronchiolar-alveolarcarcinoma. Adv Intern Med 1960; 10:329–358; Thomas et al., Br J DisChest 1985; 79:132–140; Bennett et al., Cancer 1969; 24:876–887).Recently, there have been several reports of an increase in theincidence of bronchioloalveolar carcinomas. Among patients included inseven Lung Cancer Study Group protocols between 1977 and 1988, 14.5% metthe classification criteria for bronchioloalveolar carcinoma (Grover etal., Ann Surg 1989; 209:779–90). In a 1988 study (Gazdar et al.,. SemOnc 1988; 15:215–225), the authors noted that bronchioloalveolarcarcinoma was being diagnosed with increasing frequency in theWashington-Baltimore area. In a study of 505 autopsied cases of lungcancer diagnosed between 1973 and 1989 in New Jersey, other authors(Auerbach et al., Cancer 1991; 68:1973–1977) found that the incidence ofbronchioloalveolar carcinoma increased from 9.3% to 20.3%, more than anyother cell type. A similar increase was reported in Japan (Ikeda et al.,Lung cancer 1991; 7:157164). For the time period 1982–1985, 71% of alladenocarcinomas were classif as a the bronchioloalveolar subtype. It isnot clear how much, if any, of the recent increases observed for BAC canbe explained by changes in diagnostic criteria.

While lung cancer typically shows a strong male predominance, themale/female ratio varies with histologic type. The male/female incidenceratios vary from a high for squamous cell carcinoma (M/F=2.4) to a lowfor adenocarcinoma (M/F=1.4) (Anton-Culver et al., Cancer Res 1988;48:6580–6583). The male/female ratio for bronchioloalveolar carcinomahas been reported to range from that observed in all adenocarcinomas toless than unity. Two studies (Bennett et al., Cancer 1969; 24:876–887;Ikeda et al., Lung cancer 1991; 7:157164) reported a lesser malepredominance for bronchioloalveolar carcinoma compared to all lungcancers but similar to that for all adenocarcinomas. Among patientsincluded in the Lung Cancer Study Group protocols, a male/female ratioof 1.4 was observed for bronchioloalveolar carcinoma compared to 1.7 forother adenocarcinomas (Grover et al., Ann Surg 1989; 209:779–90).Similarly, the male predominance among SEER cases was less forbronchioloalveolar carcinoma (1.2) than for other adenocarcinomas (1.8)(Percy et al., Lung cancer: causes and prevention. Mizell M, Correa P,eds. Verlag Chemie International Inc. 1984). Another study (Schraufnagelet al., Am Rev Resp Dis 1982; 125:7479) however, reported an excess ofbronchioloalveolar carcinoma in females compared to males. The malepredominance for bronchioloalveolar carcinoma is clearly less than forother types of lung cancer and evidence suggests that it is close to,and possibly less than, unity. Studies of lung cancer cell typesconsistently report a younger age at diagnosis for adenocarcinoma(including bronchioloalveolar carcinoma) than for other cell types inboth men and women (Anton-Culver et al., Cancer Res 1988; 48:6580–6583;McDuffie et al., J Clin Epidmiol 1991; 44:537–544; Greenberg et al.,JNCI 1984; 72:599–603; Tsai et al., Cancer Det Prev 1988; 11:235–238).Few studies have compared the age distribution for cases ofbronchioloalveolar carcinoma alone with other cell types. Two studies(Liebow, Bronchiolar-alveolar carcinoma. Adv Intern Med 1960;10:329–358; Storey et al., J Thorac Surg 1953; 26:331–403) describedbronchioloalveolar carcinoma as occurring with higher frequency inyounger ages compared to other cell types. Another study (Schraufnagelet al., Am Rev Resp Dis 1982; 125:74–79) noted the same age distributionfor bronchioloalveolar carcinoma and other adenocarcinomas but youngerthan for squamous cell carcinomas. Contrary to these reports, the LungCancer Study Group (Grover et al., Ann Surg 1989; 209:779–90) reportedthat bronchioloalveolar carcinoma occurs more frequently in olderpatients than does adenocarcinoma. It is not clear at this time whetheror not the age distribution for bronchioloalveolar carcinoma differsfrom other adenocarcinomas. Survival for bronchioloalveolar carcinomacompared favorably with other adenocarcinomas and large cell carcinomain the Lung Cancer Study Group experience (Grover et al., Ann Surg 1989;209:779–90). Survival rates during the first two years after diagnosiswere also better than for squamous cell carcinoma, although survivalrates were equal after two years.

Adenocarcinoma of the lung has been shown to be associated withcigarette smoking but not as strongly associated with smoking assquamous or small cell carcinomas (McDuffie et al., Cancer 1987;59:1825–30; Anton-Culver et al., Cancer Res 1988; 48:6580–6583; Osann etal., Cancer Res 1991; 51:4893–4897; Schoenberg et al., Am J Epidemiol1989; 130:688–695). Risk ratios for adenocarcinoma associated withcigarette smoking in New Jersey men and women were 4.8 and 3.6respectively (Schoenberg et al., Am J Epidemiol 1989; 130:688–695).Relative risks have not been calculated for bronchioloalveolarcarcinoma. One study (Ikeda et al., Lung cancer 1991; 7:157164) noted asimilar rate of tobacco use among cases of bronchioloalveolar carcinomaas for the overall group of adenocarcinomas. Others reported a higherproportion of non-smokers among cases of bronchioloalveolar carcinomathan among other adenocarcinomas (Schraufnagel et al., Am Rev Resp Dis1982; 125:74–79; Auerbach et al., Cancer 1991; 68:1973–1977) and lowermean pack-years of exposure to cigarettes compared to other cell types(Schraufnagel et al., Am Rev Resp Dis 1982; 125:7479). The Lung CancerStudy Group (Grover et al., Ann Surg 1989; 209:779–90) reported thatpatients with bronchioloalveolar carcinoma were significantly lesslikely to have a history of smoking than were other cases ofadenocarcinoma. While it is clear that bronchioloalveolar carcinoma isat most weakly associated with smoking, it is not clear whether theassociation with smoking differs from that observed for otheradenocarcinomas. Because the association with smoking is weak at best,smoking is an unlikely explanation for the recent increases observed.

Exposure to sidestream or secondhand smoke in the home and workplacehave been shown to double the risk of lung cancer in non-smokers(Janerich et al., New Engl J Med 1990; 323:632–6). Passive smokingsignificantly increased risk for adenocarcinoma in a study of Chinesewomen (Lam et al., Br J Cancer 1987; 55:673–8). However, no significantincrease in risk for adenocarcinoma with passive smoking was noted inanother study (Wu et al., JNCI 1985; 74:747–751). An increase in lungcancer risk associated with exposure to cooking oil vapors has also beenreported (Gao et al., In J Cancer 1987; 40:604609). The importance ofpassive smoke for bronchioloalveolar carcinoma has not been wellstudied.

Incidence of bronchioloalveolar carcinoma is increased in patients withscleroderma (Montgomery R D, Stirling G A, Hamer N A J. Bronchiolarcarcinoma in progressive systemic sclerosis. Lancet 1964; 1:586–7) andis associated with parenchymal scarring and interstitial inflammation ofthe lung (Liebow “Bronchiolar-alveolar carcinoma” Adv Intern Med 1960;10:329–358; Marcq et al, Am Rev Respir Dis 1973; 107:621–629). Although60% of bronchioloalveolar carcinomas in one study had radiographicevidence of a prior lesion in the same location as the tumor, no casesgave a medical history of illness including emphysema, tuberculosis,pneumonia, or pulmonary thromboembolism which could have caused scarring(Schraufnagel et al., Am Rev Resp Dis 1982; 125:74–79). Experimentalevidence suggests that the scarring may be the result of the cancerrather than the cause (Barsky et al., Am J Pathol 1986; 124:412–419).The Lung Cancer Study Group reported that patients withbronchioloalveolar carcinoma were less likely than other adenocarcinomapatients to have a history of chronic lung disease (Grover et al., AnnSurg 1989; 209:779–90). However, this variable was highly correlatedwith smoking. Contamination of indoor air by radon from soil, water, orbuilding materials has been shown to be a potentially important cause oflung cancer (Samet J M., JNCI 1989; 81:745–757). Exposure to radon inhomes appears to carry only a small increase in risk for non-smokers(Svensson et al., Cancer Res 1989; 49:1861–1865). When risks wereestimated by histologic type, this study (Svensson et al., Cancer Res1989; 49:1861–1865) also noted that adenocarcinoma had the lowestincrease in risk associated with radon exposure in homes of any celltype. Small cell carcinoma has most frequently been reported as thepredominant type of lung cancer in uranium miners exposed to radon(Samet J M., JNCI 1989; 81:745–757). Although there have been no reportsof an increase in bronchioloalveolar lung cancer in uranium miners,contamination of indoor air could contribute to an excess of this celltype in women who are more likely to spend time at home. Occupationalrisk factors for bronchioloalveolar carcinoma have not been identified.One study (Schraufnagel et al., Am Rev Resp Dis 1982; 125:74–79) foundno clear predominance of any occupational group in his series ofbronchioloalveolar lung cancer cases. Squamous and small cell carcinomashave most frequently been associated with occupational exposures.However, an increase in the proportion of adenocarcinomas has been notedwith exposure to asbestos, beryllium, and polyvinyl chloride (Ives etal., Am Rev Respir Dis 1983; 128:195–209). Because of the equalfrequencies of this cancer in men and women, and because occupationalexposures are more common in men than in women, it is unlikely thatoccupation is responsible for the recent increase in this disease.

Because cats and dogs get tumors that clinicopathologically resembletheir human counterparts yet presumably are not exposed to the sametypes of exogenous carcinogenic factors, a study of their molecularalterations might be revealing. Bronchioloalveolar lung carcinoma (BAC)is an example of such a cancer which involves the lungs diffusely in allthree species.

Unlike other forms of lung cancer, BAC naturally occurs in two non-humanspecies: sheep and cats. Neither of these animals are exposed to mainstream or second hand smoke. Human BAC closely resembles histologicallyan infectious endemic disease of sheep called jaagsiekte (Bonne et al.,Am J Cancer 1939; 35:491–501; De la Heras et al., Eur Respir J. 2000Aug.;16(2):330–2. The sheep form occurs as ovine pulmonary adenomatosisor jaagsiekte, a disease caused by an exogenous retrovirus (JSRV). Insheep, the disease manifests as a diffuse pulmonic adenomatosis commonlycalled SPA for short. The SPA complex is of particular interest, since:at least some, and probably all, forms are infectious; members of theSPA complex range in pathology from inflammatory or infiltrative tocarcinomatous; and various forms of SPA, while almost certainly of viraletiology, can occur in both epizootic and enzootic form, thus resemblingboth the picture of classic viral transmission (epizootic) and that ofnatural tumor epidemiology (occurring at low rates, or enzootic) inwhich viruses are not ordinarily thought to be implicated.Retrovirus-induced pulmonary carcinoma in sheep was achievedexperimentally about a decade ago (DeMartini et al., JNCI 1987;79:167–177). The cell of origin that gives rise to sheep jaagsiekte isthought to be the Type II pneumocyte, a cell thought also to give originto one subtype of human non-mucinous BAC. The exogenous virus thoughtresponsible for jaagsiekte has been cloned and reliably distinguishedfrom endogenous retroviral sequences (Palmarini et al., J of GeneralVirology 1995: 76: 2731–2737; Palmarini et al., J of Virology 1996; 70:1618–1623; Palmarini et al., J of Gen Virology 1996a; 77: 2991–2998; Baiet al., J of Virology 1996; 70: 3159–3168). The feline form naturallyoccurs in old (>10 years) pure bred Persian and Himalayan cats. The catform of BAC has not been at all studied and is not endemic norcontagious. It occurs sporadically and spontaneously in older pure bredcats, especially Persian and Himalayan. With respect to its sporadic andspontaneous nature, it has more similarities to human BAC than doessheep BAC (jaagsiekte). Feline BAC is also thought to be of Type IIpneumocyte origin.

As is known in the art, animals and humans are affected by a variety ofrelated and/or common pathogens. For example cats are frequentlyinfected with or are carriers of toxoplasmosis, an infectious organismwhich can seriously harm the developing human fetus in pregnant women.It has been difficult to grow toxoplasmosis in culture and develop avaccine against this disease. But since cats are the natural host forthis organism, it has been suggested that feline cells may provide anurturing environment for growing organisms such as toxoplasmosis invitro. Consequently, there is a need in the art for reagents such asmammalian cell lines that can be used in the examination of mammalianpathogens. In addition, there is a need in the art for reagents such asmammalian cell lines that can be used in the examination of mammaliancancers such as BAC. The invention disclosed herein meets these needs.

SUMMARY OF THE INVENTION

The invention disclosed herein provides a model system for studyinghuman and related mammalian pathologies such as BAC. In particular, thepresent invention provides transplantable xenograft (SPARKY-X) and cellline (SPARKY) derived from a malignant pleural effusion of a 12 year-oldPersian male with autopsy-confirmed BAC. SPARKY-X exhibits a classiclepidic BAC growth pattern and stimulated angiogenesis and stroma.SPARKY exhibits a type II pneumocyte origin: lamellar bodiesultrastructurally and surfactant expression by Northern blot. SPARKY'skaryotype was aneuploid (66 chromosomes: 38=diploid). P53 showed a G toT transversion at codon 167, the feline equivalent of human codon 175,one of the many hot spots mutated in smokers. Ha-ras, Ki-ras were notaltered. In addition, as shown by RT-PCR, SPARKY expresses retroviralgag transcripts which Ore 90% identical to exogenous JSRV (jaagsiekteretrovirus), the retroviral cause of sheep BAC. The feline transcriptsof SPARKY, like the exogenous JSRV retroviral transcripts, contains aSca I site. The molecular alterations observed in SPARKY that are sharedwith sheep and human lung cancers provides evidence of an overlappingpathway of tumorigenesis. The disclosure provided herein teaches methodsin which SPARKY-X and SPARKY can be used in methods of evaluatingpathogens such as jaagsiekte-type retroviruses and Toxoplasma gondii invitro and in vivo.

As noted above, the inventions disclosed herein relate to felinetransplantable lung xenografts and cell lines. These cells exhibit anumber of unique characteristics which allows the skilled artisan to usethem in experimental models of lung cancer in order to dissect out themolecular basis of this phenotype. Moreover, this experimental model oflung cancer can be used to identify molecular targets for therapeuticintervention and to assess the efficacy of a broad spectrum ofdiagnostic and therapeutic agents.

One embodiment of the invention consists of a feline lung cancerxenograft. In a preferred embodiment the xenograft is the felinexenograft referred to as SPARKY-X deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va., USA(ATCC) on Jan. 19, 2001 and received patent deposit designationPTA-2920. A related embodiment of the disclosed invention consists of anin vitro cell line culture deposited with the ATCC on Jan. 19, 2001 andreceived patent deposit designation PTA-2919. Methods for generating thedisclosed xenografts are also described. Another embodiment of theinvention consists of methods of identifying a molecule whose expressionis modulated in lung cancer by determining the level of expression of atleast one molecule in the feline lung cancer xenograft; and comparingthis to the level of expression of the same molecule in a cell havingcharacteristics which are distinct from the feline lung cancerxenograft. In preferred embodiments of this invention, the level ofexpression of the molecule of the lung cancer xenograft is determined bymethods selected from the group consisting of: Northern blotting,Southern blotting, Western blotting and polymerase chain reaction.

Yet another embodiment of the invention consists of an animal model forlung cancer comprising an immunocompromised host animal inoculated witha feline lung cancer xenograft. In preferred embodiments of thisinvention, the host animal is a nude mouse and the xenograft is thexenograft designated SPARKY-X. Another embodiment of the inventionconsists of methods for evaluating at least one agent for treating lungcancer by utilizing a immunocompromised host animal inoculated with afeline lung cancer xenograft, administering at least one agent to saidinoculated immunocompromised host animal and evaluating the effects ofthe agent(s) on the feline lung cancer xenograft. Optionally, the agentthat is being evaluated targets a molecule that is identified as beingassociated with the oncogenic phenotype.

Another embodiment of the invention comprises a method of culturing apathogen in the disclosed feline cells by inoculating the cell line withthe pathogen and growing the pathogen under conditions known tofacilitate its growth. Optionally the pathogen is toxoplasmosis gondiiand is cultured and recovered by methods known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Low power magnification of photomicrograph of Spark's lungshows bronchioloalveolar pattern, so-called lepidic pattern of BAC.

FIG. 1B: High power magnification of photomicrograph of Sparky's lungshows a single cell layer of transformed Type II pneumocytes in abronchioloalveolar pattern.

FIG. 1C: SPARKY-X, the nude/Scid mouse xenograft derived from SPARKY,maintains a bronchioloalveolar pattern even in the mouse.

FIG. 2: A computer enhanced R-banded feline BAC cell line karyotype. Themodal number is 66. Most abnormalities are numerical. The arrowsindicate some structural aberrations including an addition of a darkband on the p-arm of an A2 chromosome, an inversion and deletion in onecopy of a C1 chromosome.

FIG. 3A: Results of JSRV gag sequencing in feline BAC showing thehomology between JSRV gag sequences in Jaagsiekte and feline BAC. Acomparison of feline BAC to exogenous shows the presence of a Sca I siteand 19 mismatched (91.7% homology). A comparison of feline BAC toendogenous shows the presence of a Sca I site and 24 mismatched (89.5%homology). The nucleotide sequences in this figure include EXO SPA (SEQID NO: 11), ENDO SPA (SEQ ID NO: 12), and feline BAC (SEQ ID NO: 13).

FIG. 3B: Results of amino add comparison in feline BAC showing the aminoadd fidelity of JSRV gag transcripts (SEQ ID NO: 19) in Jaagsiekte andfeline BAC (F-BAC).

FIG. 4A: Results of JSRV gag sequencing in human BAC showing homologybetween jsrv gag sequences in a jaagsiekte and five cases of humanBAC/PAC and one case of feline BAC. The nucleotide sequences in thisfigure include EXO SPA (SEQ ID NO: 11), ENDO SPA (SEQ ID NO: 12), felineBAC (SEQ ID NO: 13), and human BAC 1–human BAC 5 (SEQ ID Nos: 15–18respectively).

FIG. 4B: Results of amino acid comparison in human BAC showing the aminoacid fidelity of JSRV gag transcripts (SEQ ID NO: 19 in jaagsiekte,human BAC/PAC and feline BAC.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/ox for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin see Ausubel et al., Current Protocols in Molecular Biology, WileyInterscience Publishers, (1995) and Sambrook et al., Molecular Cloning:A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. As appropriate, procedures involving theuse of commercially available kits and reagents are generally carriedout in accordance with manufacturer defined protocols and/or parametersunless otherwise noted.

Embodiments of the invention disclosed herein relate to feline lungcarcinoma xenografts (SPARKY-X) and cell lines (SPARKY). The cells,animal models and assays disclosed herein allow one to analyze thecellular and molecular mechanisms associated with the oncogenesis ofcells of the bronchioloalveolar lineage, which will lead to bettermethodological tools for diagnosing and treating diseases such as lungcancers. For example, as described in detail below, SPARKY and SPARKY-Xexhibit a number of unique characteristics which allows the skilledartisan to use them in experimental models of lung carcinoma to, forexample, dissect out the molecular basis of this phenotype. Moreover,these experimental models of lung carcinoma can be used to identifymolecular targets for therapeutic intervention and to assess theefficacy of a broad spectrum of diagnostic and therapeutic agents. Theinvention further provides methods of culturing pathogens in thedisclosed feline cells by inoculating the cells with the pathogen andgrowing the pathogen under conditions known to facilitate its growth.Optionally the pathogen so cultured can be recovered by methods known inthe art. Certain methods and reagents of the invention are related toPCT International Application No. PCT/US00/25299, published on Mar. 22,2001 under International Publication No. WO 01/19967, which isincorporated herein by reference.

Establishing Feline BAC Xenografts and BAC Cell Lines

A 12 year old Persian cat, named Sparky presented with shortness ofbreath and was found to have bilateral pleural effusions which containedmalignant cells on pleuracentesis. These cells were cultured and gaverise to an immortal cell line, SPARKY. In general the vast majority ofsuccessfully established human and animal cell lines have been obtainedfrom pleural effusions as was the case with SPARKY. Without being boundby specific theory, this is believed to be influenced by two things: thefact that contaminating fibroblasts are not present in pleural effusionsand are not able therefore to overgrow the culture and the fact thatmalignant cells in pleural effusions have already “adapted” to growingin a culture media of sorts. The successful establishment of such a cellline (SPARKY) and subsequent xenograft (SPARKY-X) provides novel modelsthat allow for the study of common animal-human pathogens implicated incancer and infectious disease. Embodiments of the invention that relateto establishing the feline BAC xenografts consist of obtaining cellsfrom a pleural effusion via methods known in the art (e.g. biopsy) andtransplanting them (preferably subcutaneously) in to an non-felineimmunocompromised host, allowing the cells to proliferate and thenisolating feline cells from the host. Optionally the cells are passagedin the host serially prior to their isolation and characterization.Embodiments of the invention that relate to establishing the feline BACxenografts consist of obtaining cells from a pleural effusion viamethods known in the art (e.g. needle biopsy) and placing them in tissueculture under conditions amenable to their growth, allowing them toproliferate and then isolating the cells. Optionally the cells arepassaged in the culture serially (e.g. serial dilution to establishclonal populations) prior to their isolation and characterization.

Characterizing Feline BAC Xenografts and BAC Cell Lines

Sparky the cat was found to have PAC/BAC with extensive lobarinvolvement. Sections of Sparky's lung revealed extensive replacement ofpulmonary parenchyma by BAC which grew in a lepidic pattern alongalveolar septae (FIG. 1A; FIG. 1B). The cell line from the malignantpleural effusion has been successfully passed over 30 times, grows as amonolayer in cell culture and exhibits large secretory cytoplasmicvacuoles. The cell line when injected into nude and Scid (SPARKY-X) miceis fully tumorigenic and forms nodules which exhibit a lepidic (BAC)growth pattern even when injected subcutaneously (FIG. 1C). Thisprovides evidence that the lepidic or alveolar growth patterncharacteristic of BAC is an inherent property of the transformed Type IIpneumocyte and is apparently not related to the presence of pre-existingalveolar spaces within lung parenchyma. The cell line ultrastructurallycontains lamellar bodies and exhibits by Northern blot surfactanttranscripts. Sparky's tumor, the derived cell line (SPARKY) and thederived xenograft (SPARKY-X) all showed intense immunoreactivity to APOA1, a monoclonal antibody to surfactant. SPARKY was further studied forproof of feline origin with feline specific genomic probes and proof ofunique identity with DNA fingerprinting. SPARKY was also studied withflow cytometry and a detailed karyotype analysis. SPARKY was alsostudied for specific mutations in key genes by PCR followed bysequencing.

SPARKY's genome is clearly feline and exhibits a unique DNA fingerprint.SPARKY was aneuploid by flow cytometric examination and also aneuploidby detailed karyotype analysis (66 chromosomes: 38=normal felinediploid) FIG. 2). Sparky's tumor, the derived cell line (SPARKY) and thederived xenograft (SPARKY-X all showed intense immunoreactivity tomonoclonal antibodies to p53. PCR-sequencing analysis of the p53 andHa-ras and Ki-ras genes showed a G to T transversion at codon 167(Arginine→Leucine), the feline equivalent of human codon 175, one of themany hot spots mutated in lung cancers of smokers. Ha-ras and Ki-raswere not altered.

Interestingly, as shown in FIG. 3A, SPARKY expressed by RT-PCRretroviral gag transcripts which were 91.7% identical to exogenous JSRV(jaagsiekte retrovirus), the retroviral cause of sheep BAC (for adescription of jaagsiekte retrovirus, see, e.g. Palmarini et al., J NatlCancer Inst 2001 Nov. 7;93(21):1603–14; Palmarini et al., TrendsMicrobiol. 1997 Dec.;5(12):478–83; DeMartini et al., Vet Clin North AimFood Anim Pract. 1997 Mar.;13(1):55–70; and Hecht et al., Br Vet J. 1996Jul.;152(4):395409). None of the base pair differences in thesetranscripts resulted in an amino acid substitution (FIG. 3B). The felinetranscripts of SPARKY, like the exogenous JSRV retroviral transcripts,contained a Sca I site (FIG. 3A). Although feline BAC nay have adifferent etiology, the molecular alterations it shares with sheep andhuman lung cancer provide evidence of an overlapping pathway oftumorigenesis.

Immune Deficient Animal Hosts For Practicing Embodiments of theInvention

Severe combined immune deficient (SCID) mice are the preferred animalhost utilized in the practice of certain embodiments of the invention.Various other immune deficient mice, rodents or animals may be used,including those which are deficient as a result of a genetic defect,which may be naturally occurring or induced, such as, for example, nudemice, Rag 1 and/or Rag 2 mice, and the like, and mice which have beencross-bred with these mice and have an immunocompromised background. Thedeficiency may be, for example, as a result of a genetic defect inrecombination, a genetically defective thymus or a defective T-cellreceptor region. Induced immune deficiency may be as a result ofadministration of an immunosuppressant, e.g. cyclosporin, removal of thethymus, etc. Various transgenic immune deficient mice are currentlyavailable or can be developed in accordance with conventionaltechniques. Ideally, the immune deficient mouse will have a defect whichinhibits maturation of lymphocytes, particularly lacking the ability torearrange the T-cell receptor region. In one embodiment, C17 scid/scidmice are used. In addition to mice, immune deficient rats or similarrodents may also be employed in the practice of the invention.

Xenograft Animal Models that Simulate Lung Cancer

Embodiments of the invention provide murine xenograft models whichsimulate or mic bronchioloalveolar carcinoma from primary tumorformation. Also provided are methods for propagating bronchioloalveolarcarcinoma tissue as subcutaneous xenografts in immune deficient mice. Inthe practice of the invention, bronchioloalveolar carcinoma xenograftsmay be established in immune deficient mice by the subcutaneousimplantation of fresh bronchioloalveolar carcinoma explants surgicallyremoved from mammals with bronchioloalveolar carcinomas. The site ofimplantation may be into any subcutaneous site which will permit bloodsupply to reach the implant, such as the flanks of the host animal.Tissue from primary bronchioloalveolar carcinomas as well as from sitesof lymph node, lung, bone, and other organ metastases may be used toestablish the bronchioloalveolar carcinoma xenografts of the invention.Bronchioloalveolar carcinoma explants may be introduced in conjunctionwith a basement membrane composition, such as Matrigel (U.S. Pat. No.5,508,188), an extracellular matrix preparation which has been shown toenhance the growth of tumors in vivo (et al., 1993; Noel et al., 1992;Pretlow et al., 1991), as well as other similar types of compositions.Once established, the xenograft tumors grow to considerable size,providing substantial tissue volumes for further use. Xenografts of theinvention retain the clinical phenotype as determined by growthcharacteristics reflective of the clinical situation.

This and other aspects of the invention described herein provide toolsfor studying the pathogenesis and treatment of bronchioloalveolarcarcinoma. For example, immune deficient mice bearing subcutaneous (andother) xenografts may be used to evaluate the effect of variousbronchioloalveolar carcinoma treatments (e.g., therapeutic compositions,gene therapies, immunotherapies, etc.) on the growth of tumors andprogression of disease. Xenograft cells may be used to identify novelgenes and genes which are differentially expressed in bronchioloalveolarcarcinoma cells, or to analyze the effect such genes have on theprogression of bronchioloalveolar carcinomas. For example, the geneticcompositions of bronchioloalveolar carcinoma cells from xenograftshaving differing characteristics (e.g. rates of growth and/oraggressiveness) may be compared to each other as well as to the geneticcompositions of normal bronchioloalveolar cells. Likewise, the geneticcompositions of metastatic bronchioloalveolar carcinoma cells may becompared to those of nonmetastatic bronchioloalveolar carcinoma cells.Various nucleic acid subtraction and sampling techniques may be used forthis purpose, including, for example, representational differenceanalysis (RDA). In addition, bronchioloalveolar carcinoma xenograftcells may be used for the introduction of various genetic capabilities,including the introduction of various genes, antisense sequences,ribozymes, regulatory sequences which enhance or repress the expressionof endogenous genes, and so forth.

As discussed in detail below, this aspect of the invention also providesassays for determining the function or effect of various genes onbronchioloalveolar carcinoma cells. In one embodiment, the assaycomprises isolating bronchioloalveolar carcinoma cells from abronchioloalveolar carcinoma xenograft (e.g., subcutaneous or i.p.),transducing the cells with the gene of interest such that the transducedcells express or overexpress the gene, establishing a xenograft tumor ina SCID or other immune deficient mouse with the transduced cells, andevaluating the growth of the resulting xenograft. The effect ofexpressing the gene on the growth of the xenograft may be determined byreference to a control xenograft established with untransducedbronchioloalveolar carcinoma cells, preferably isolated from the sameparental xenograft. In another embodiment, the assay comprisesgenerating a bronchioloalveolar carcinoma xenograft, transducing thecells of the xenograft with the gene of interest in vivo, and evaluatingthe growth of the xenograft, wherein the effect of the gene on thegrowth of the xenograft may be determined by reference to a controlxenograft.

Similarly, the invention provides assays for determining the effect ofcandidate therapeutic compositions or treatments on the growth ofbronchioloalveolar carcinoma cells. In such contexts, one or morepotential therapeutic agents (typically in a composition including apharmaceutically acceptable carrier) such as chemotherapeutic compounds,antiangiogenic molecules, and known or potential modulators of BACgrowth and/or differentiation are administered to the xenograft model(e.g. nude mice harboring the xenograft) and the effects of the agent ontumor growth, metastasis, differentiation etc. are examined andcharacterized. In addition to examining various therapeutic agents, onecan examine the effects of a specific treatment or treatments such as aspecific therapeutic regimen (e.g. a specific combination of agents, aspecific mode of administration, specific time period or periods oftreatment etc.) in an analogous manner. In such embodiments of theinvention, the control typically consists of an equivalent xenograftmodel that has not been exposed to the agent or regimen. In oneembodiment, an assay comprises applying the agent composition ortreatment to a SCID mouse or other immune deficient mammal bearing asubcutaneous bronchioloalveolar carcinoma xenograft and determining theeffect of the treatment on the growth of the xenograft.

Agent compositions for use in the above mentioned embodiments of theinvention can be prepared by mixing the desired molecule having theappropriate degree of purity with optional pharmaceutically acceptablecarriers, excipients, or stabilizers (Remington's PharmaceuticalSciences, 16th edition, Osol, A. ed. (1980)), in the form of lyophilizedformulations, aqueous solutions or aqueous suspensions. Examples ofroutes of administration include parenteral, e.g., intravenous,intradermal, intramuscular, subcutaneous, oral (e.g., inhalation)transdermal (topical), transmucosal (e.g. a nasal spray), and rectaladministration. The agent may also be administered by perfusiontechniques, such as isolated tissue perfusion, to exert localtherapeutic effects. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution; fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as EDTA; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. Regimens of administration may vary. Asingle dose or multiple doses of the agent may be used. Such regimenscan vary depending on the severity of the disease and the desiredoutcome. Following administration of an agent to the mammal, themammal's physiological condition can be monitored in various ways wellknown to the skilled practitioner familiar with BAC.

Embodiments of the invention also have other clinical applications,including using the model in a method to assess prognosis of a patientwith bronchioloalveolar carcinoma. For example, in one embodiment, themethod comprises implanting a tumor sample from the patient into animmune deficient mouse subcutaneously, and allowing the implanted sampleto grow as a xenograft in the mouse. The rates of xenograft growth maybe used as a prognostic indicator. The results of such analysis mayassist a treating oncologist in determining how aggressively to treat apatient.

Analytical Methods for Characterizing Bronchioloalveolar Carcinomas

A wide variety of analytical and comparative methodologies forcharacterizing bronchioloalveolar carcinomas such as the xenograftsand/or cell lines disclosed herein are known in the art. For example,gene expression in a xenograft and/or cell line may be measured in asample directly, for example, by conventional Southern blotting,Northern blotting to quantitate the transcription of mRNA (Thomas, Proc.Natl. Acad Sci. USA, 77:5201–5205 (1980)), dot blotting (DNA analysis),or in situ hybridization, using an appropriately labeled probe, based onthe sequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Alternatively, gene expression may be measured by immunological methods,such as immunohistochemical staining of cells or tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. Antibodies useful for immunohistochemicalstaining and/or assay of sample fluids may be either monoclonal orpolyclonal, and may be prepared in any mammal. Conveniently, theantibodies may be prepared against a native sequence polypeptide oragainst a synthetic peptide based on the DNA sequences provided hereinor against exogenous sequence fused to DNA and encoding a specificantibody epitope.

A number of illustrative methods are known in the art that can be usedto analyze bronchioloalveolar xenografts and cell lines. For example DNAprofiling can be used to distinguish murine from feline DNA andquantitate the murine DNA component of the xenografts, to comparecancerous versus noncancerous bronchioloalveolar cells and to comparevarious bronchioloalveolar cancer phenotypes (e.g. Aggressive versusnonagressive). Western blot and zymography analysis can be utilized tocompare SPARKY-X with non-xenografts with respect to candidate effectormolecules. Immunocytochemistry and FISH can also be utilized for thispurpose.

In addition, the invention disclosed herein allows one to analyze thepathological features of cancer cells of the bronchioloalveolar lineage,which will lead to better therapies aimed at preventing diseaserecurrences. In this context, a variety of analytical and comparativemethodologies for characterizing the pathological features of cancercells of the bronchioloalveolar lineage are known in the art.

Methods for Identifying Molecules Associated with the MalignantPhenotype

The invention disclosed herein allows a variety of molecular comparisonto identify those genes that are uniquely expressed (or not expressed)by bronchioloalveolar carcinomas to see whether they are associated withoncogenesis and/or the malignant phenotype/genotype. Becausebronchioloalveolar carcinoma is a unique disease, unique upstream genesregulate the expression of those effector molecules which cause itsphenotype. Therefore one can use methods known in the art to identifythe higher level genes which regulate the oncogenic phenotype. Inidentifying the upstream genes that are uniquely expressed (or notexpressed) by bronchioloalveolar carcinomas, one can also investigatewhether they trigger separate or common downstream events linked to thispathology.

The xenograft model disclosed herein allows for a variety of differentapproaches to be used to identify the genetic and phenotypic basis ofbronchioloalveolar carcinomas. As discussed in detail below, preferablythese approaches involve a comparison of SPARKY-X or SPARKY tononcancerous bronchioloalveolar cells, preferably those of the type IIpneumocyte lineage. For example, the invention provides a large numberof methods for identifying one or more molecules whose expression ismodulated in bronchioloalveolar cancer by determining the level ofexpression of at least one molecule in a mammalian (e.g. feline)bronchioloalveolar cancer xenograft; and comparing this to the level ofexpression of the same molecule in a cell having characteristics whichare distinct from the human bronchioloalveolar cancer xenograft (e.g.non cancerous bronchioloalveolar cells). In preferred embodiments ofthis invention, the level of expression of the molecule of thebronchioloalveolar cancer xenograft is determined by a method selectedfrom the group consisting of: Northern Blotting, Southern Blotting,Western Blotting and polymerase chain reaction.

One exemplary approach involves Northern and Western blot comparisons ofSPARKY-X to non cancerous bronchioloalveolar cells. As the class ofmolecules which mediate the phenotype are likely to be either moleculesof the adhesion family (either on tumor cells or endothelial cells),growth and differentiation factors (expressed by cancerous cells) orproteolytic enzymes elaborated by tumor cells which facilitate growthone can conduct a comparative screen of the major effector moleculespreviously implicated in the above mentioned processes (e.g. p53 andk-ras as disclosed herein).

Yet another approach is a mRNA shotgun comparison using microarraylibraries. One can then utilize microarray gene chips containing bothcloned human or feline secretory molecules and SSTs (secreted sequencetags) which are constructed from a signal trap selection strategy (seee.g. Honjo et al., Science 268: 600–603, (1993)). In this context humanand murine secretory microarray chips are available commercially.Therefore, one can carry out a mRNA shotgun comparison of SPARKY-X withnon cancerous bronchioloalveolar cells to look for targets that aredifferentially expressed in SPARKY-X as compared to non-cancerous cells.

Yet another approach involves in vivo subtraction strategies such asthose using expanded human recombinant phage libraries. One suchapproach comprises an in vivo subtraction strategy using expanded humanrecombinant phage libraries injected into the tail vein of miceharboring SPARKY-X with the anticipated recovery of Ig-phages thatselectively bind to SPARKY-X. The power of the immune system stems fromits ability to diversify antigen receptors. In the case of B cells, DNArearrangement combinatory events (i.e., random pairing of heavy andlight chains) and specialized “diversity-producing” mechanisms (e.g., Nand P nucleotide additions) produces an antibody repertoire of 10⁶unique molecules. A similar repertoire complexity can be obtained withIg-phage libraries. Diversity in such libraries can be achieved by theproduction of semi-synthetic Ig-phage libraries (see e.g. Griffiths etal., EMBO J. 13: 3245–3260 (1994). Semisynthetic phage libraries arecreated by introducing mutations via error prone polymerase, byreshuffling heavy and light chains, or by randomly mutating the heavychain complementarity determining region 3 (CDR3) of the Ag bindingsite. Such manipulation can produce library sizes dose to 10¹³. Recentstudies have demonstrated the feasibility of injecting a phage libraryinto the tail veins of mice in which selected clones immunolocalize andare able to be recovered from cells such as xenografts (see e.g. Arap etal. Science 279: 377–380 (1998)). This approach is especially suited forour xenograft model because SPARKY-X is present in pleural effusions andhence the injected Ig-phage clones would have easy access to cancerassociated antigens. An in vivo subtraction strategy in mice harboringSPARKY-X is preferable because it will eliminate those phages which bindeither non-specifically to tumor vasculature or to generally presentbronchioloalveolar carcinoma surface antigens or receptors that havenothing to do with the carcinoma phenotype. Following tail veininjection SPARKY-X can be extirpated and bound phages recovered,propagated and re-injected for subsequent rounds of in vivo selectionwith the ultimate recovery of Ig-phages that selectively bind to surfacedeterminants on either the bronchioloalveolar carcinoma cells or theendothelial cells within SPARKY-X that specifically mediate itsphenotype. The cloned phages can be used to identify the surfacemolecules involved.

The approaches enumerated herein can identify candidate molecules thatmay play a role in bronchioloalveolar cancers. Once a molecule isidentified, one can transduce any candidate molecule which shows promiseinto non cancerous bronchioloalveolar cells to observe their effect onthe phenotype. For example, a nucleic acid (e.g., cDNA or genomic DNA)encoding a candidate molecule may be inserted into a replicable vectorfor cloning (amplification of the DNA) or for expression. Variousvectors are publicly available. The vector may, for example, be in theform of a plasmid, cosmid, vital particle, or phage. The appropriatenucleic acid sequence may be inserted into the vector by a variety ofprocedures. In general, DNA is inserted into an appropriate restrictionendonuclease site(s) using techniques known in the art. Vectorcomponents generally include, but are not limited to, one or more of asignal sequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.Construction of suitable vectors containing one or more of thesecomponents employs standard ligation techniques which are known to theskilled artisan.

Conversely, through an antisense technology approach one can knock out amolecule of interest that has been identified in SPARKY-X to see if thisabolishes its phenotype. Antisense technology entails the administrationof exogenous oligonucleotides which bind to a target polynucleotidelocated within the cells. The term “antisense” refers to the fact thatsuch oligonucleotides are complementary to their intracellular targets.See for example, Jack Cohen, OLIGODEOXYNUCLEOTIDES, Antisense Inhibitorsof Gene Expression, CRC Press, 1989; and Synthesis 1:1–5 (1988).Antisense oligonucleotides of the present invention include derivativessuch as S-oligonucleotides phosphorothioate derivatives or S-oligos,see, Jack Cohen, supra) which exhibit enhanced cancer cell growthmodulatory action. S-oligos (nucleoside phosphorothioates) areisoelectronic analogs of an oligonucleotide (O-oligo) in which anonbridging oxygen atom of the phosphate group is replaced by a sulfuratom. The S-oligos of the present invention may be prepared by treatmentof the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxidewhich is a sulfur transfer reagent. See Iyer, R. P. et al, J. Org. Chem.55:4693–4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc.112:1253–1254 (1990), the disclosures of which are fully incorporated byreference herein.

A variety of specific approaches for characterizing bronchioloalveolarcancer cells can be employed utilizing the methods and cells disclosedherein. For example, one can conduct a differential display analysisbetween the SPARKY-X, SPARKY and normal bronchioloalveolar cells.Preferably one will initially focus exclusively on all or nonedifferentially expressed transcripts between the SPARKYs and thenon-SPARKYs in order to identify high level regulatory genes (in theclass of oncogenes or tumor suppressor genes) as opposed to downstreamgenes in this approach. Oncogenes (e.g. by rearrangement) and suppressorgenes (e.g. by deletion) will manifest by all or none differentialexpression of these transcripts, internal consistency between SPARKY-Xand SPARKY will provide evidence that these transcripts representgenetic alterations (either amplification, rearrangement or loss),whereas divergence between SPARKY-X and SPARKY will provide evidencethat these transcripts are epigenetically rather than geneticallyregulated and likely not as important from the standpoint of possibleoncogenes or suppressor genes. All transcripts identified bydifferential display can be confirmed by Northern blot of the respectivecells cell lines and xenografts. Confirmed transcripts can be excisedfrom the differential display gel, reamplified, cloned and sequenced.Sequences can be compared with known sequences on the BLASTN, expressedsequence tag (dbEST) and The Institute for Human Genome Research (TIGR)databases.

A variety of specific and augmentary procedures for identifyingmolecules associated with the cancer phenotype are known in the art. Forexample differential display can be conducted with a third generationRNAimage Kit (GenHUnter Corporation, Nashville, Tenn.) using threeone-based-anchored oligo-dT primers to subdivide the mRNA population.With built-in restriction sites at the 5′ ends of both anchored andarbitrary 13mers, the longer primer pairs produce highly selective andreproducible cDNA patterns. This ensures that differentially expressedgenes are more readily identified, cloned and manipulated. Alternativelyone can employ laser capture microdissection. In such methods one cancollect fresh frozen cases of carcinoma and successfully microdissectthe tumor samples. One can also retrieved archived cases of severalhundred carcinomas embedded in paraffin. The availability of such tissuewill allow one to determine if the sequences (presence or absence)identified by differential display of SPARKY-X are also exhibited byactual cases of human, carcinoma. Alternatively one can utilize methodsfor the functional characterization of isolated cDNAs from differentialdisplay procedures. In such methods the role of the cDNAs can bedetermined by first isolating and characterizing their full length cDNAequivalent with RACE and ultimately determining the functional activityof these cDNAs in either SPARKY-X (for transcripts entirely absent inSPARKY-X or the non-cancerous bronchioloalveolar cells (for transcriptsexclusively present in SPARKY-X). One can initially continue thesequencing strategy described herein and perform Northern blothybridization analyses in SPARKY-X and the non-cancerousbronchioloalveolar cells. Once full length cDNAs are isolated, one canuse RNase protection assays to determine the fidelity of these cDNAs toensure that there are no mutations or alterations in these cDNAsresulting from the PCR and RACE procedures. The sequences can then besubmitted to NCBI to determine the homology of these cDNAs at thenucleotide and protein levels. The protein sequence will allows one toconsider hypotheses on the possible function and activity of the proteinas well as generate antibodies to either the purified proteins (proteinscould be generated by cDNAs in firm transcribed and translated orbacterial expressed proteins) or synthetic polypeptides. SPARKY-X andthe non-cancerous bronchioloalveolar cells can be analyzed at the RNAand protein levels to determine expression levels, cell location, andtissue distribution. SPARKY-X and the non-cancerous bronchioloalveolarcells can be transfected with expression vectors containing these cDNAsin a sense or antisense orientation to determine their effects.Specifically the effects of these cDNAs on the production or inhibitionof angiogenic factors and the induction/inhibition of angiogenesis canbe assayed in vitro and in mice respectively. Similarly the effects ofthese cDNAs on the production or inhibition of oncogenesis-relatedmolecules can be assayed in vitro and the induction/inhibition ofoncogenesis will be assayed in mice. These studies allow one todetermine the cDNA(s)' functional activity and see whether thedownstream events they trigger are related to angiogenesis orintravasation or both. If pleoitropism is demonstrated, this providesevidence that the angiogenic and oncogenic phenotypes are on the basisof the same upstream genotype and therefore related. On the other handif oncogenesis and its related gene products and angiogenesis and itsrelated effector molecules are separately regulated, this providesevidence that these two phenotypes are distinct.

Using Feline BAC Xenograft and Cell Line Models to Culture andCharacterize Common Animal-Human Pathogens Including Those Implicated inOncogenesis

The disclosure herein of established feline xenografts and cells linesthat harbor a Jaagsiekte-type tetrovirus provides novel methods for theculture and/or characterization of this virus. In addition, as felinesand humans share a group of pathogens (e.g. pasteurella, the pathogensof the so-called cat-scratch disease, trichophyton and microsporumspecies, toxoplasmosis, Cryptosporidium parvum and orthopox virusesetc.), the established feline xenografts and cells lines providepotential culture mediums for common pathogenic agents as well. Forexample, the parasite Toxoplasma gondii is used as one illustrativeagent that can be cultured in the disclosed SPARKY cells.

In addition, the disclosure herein of established feline xenografts andcells lines that harbor a Jaagsiekte-type retrovirus provides novelmethods, such as those designed to characterize this virus. For example,the disclosure provides evidence that an exogenous retrovirus isinvolved in either the initiation and/or promotion of human PAC/BAC;that the activation and expression of an endogenous retrovirus and thegene products of this tetrovirus, e.g., gag in human PAC/BAC is animportant step in the pathway of tumorigenesis analogous to theactivation of oncogenes; and that the mechanism of oncogenesis in humanPAC/BAC reactivates retroviral transcripts as downstream events of theneoplasia. These downstream events may have nothing to do withtransformation or progression per se but may reflect the end point of acommon pathway present in both human PAC/BAC, feline BAC and sheepjaagsiekte (since in jaagsiekte there is also reactivation of endogenousgag). The successful establishment of the first known feline cell lineand xenograft of BAC (SPARKY and SPARKY-X) therefore provides a meansand model to investigate these possibilities by first determiningwhether we are dealing with an exogenous or endogenous retrovirus: withthe former, attempting to recover the retrovirus from SPARKY/SPARKY-Xeither by vital particle purification or molecular cloning; with thelatter, studying the growth-promoting properties of the endogenousretrovirus and its gene products, e.g., gag, and finally detecting transfactors in feline BAC which activate endogenous retroviraltranscription. The findings using SPARKY/SPARKY-X have direct relevanceto human BAC/PAC.

The disclosure herein teaches that Jaagsiekte type retrovirus can bepresent in mammalian BAC cells. In addition, as noted herein, human BACclosely resembles histologically an infectious endemic disease of sheepcalled jaagsiekte. Consequently, embodiments of the invention include amethod of identifying a factor associated with a BAC phenotype byexamining a BAC sample for the presence of a transcript that hybridizesunder stringent conditions to a mRNA transcript encoding an amino acidsequence shown in FIG. 4. In addition, the invention disclosed hereinprovides antibodies to proteins such as JSRV-CA and the JSRV amino acidsequences disclosed herein (e.g. FIG. 4).

I. Studies of the Jaagsiekte Retrovirus, JSRV

Previous studies out of the Moredun Research Institute, Scotland, onsheep pulmonary adenomatosis (jaagsiekte), a contagious lung tumour,have indicated that the probable etiological agent is a previouslyuncultivable type D retrovirus. These studies have succeeded indeveloping a rapid transmission model for this turnout and produced thefirst and only permanent jaagsiekte cell lines. Additional studies haveclarified the role of retroviruses in contagious tumors of both sheepand goats. These studies have demonstrated unequivocally that anexogenous retrovirus plays a major role in the etiology of jaagsiekteand demonstrated related retroviruses in enzootic nasal tumors of bothsheep and goats. These related retroviruses are associated with similarcontagious tumors arising from secretory epithelial cells in therespiratory tract and may yield new clues to the mechanisms ofoncogenesis in epithelial cells. In cloning the jaagsiekte retrovirus(JSRV), the studies consistently demonstrated that it is important todistinguish the exogenous retrovirus from endogenous retroviralsequences which can also be detected by RT-PCR in normal tissues ofsheep. Exogenous retrovirus can be distinguished from these endogenousretroviral sequences in two ways: the exogenous viral transcriptscontain a novel ScaI restriction site in a gag gene which is absent inthe endogenous sequences and the exogenous retrovirus can be detectedwith a blocking enzyme-linked immunoadsorbent assay (B-ELISA). Thisassay depends on the presence of JSRV to inhibit the binding betweenpurified βgal-CA (a fusion protein containing a major capsid protein ofJSRV) and the rabbit antiserum to JSRV-CA.

Although human endogenous retroviruses or human endogenous proviral DNAshave been identified in human genomic DNA, this is the first time thatJSRV gag has been detected and in the form of an RNA transcriptexpressed in human and feline BAC. Given that JSRV is the cause of sheeppulmonic adenomatosis or jaagsiekte, a disease with strong histologicaland biological resemblance to both human and feline BAC, the discoveryof JSRV gag transcription is highly significant. This new finding andthe use of our feline BAC model serves as the basis for a number ofresearch strategies, including those described below.

Using methods known in the art, one can determine if the retroviral gagtranscripts represent an exogenous or an endogenous retrovirus. It canbe reasoned that since the sequences of our gag transcripts varied a bitfrom human individual cases of PAC/BAC and feline BAC, this suggeststhat we are dealing with an exogenous retrovirus. An endogenousretrovirus would be expected to be conserved and not vary from case tocase. However another possibility is that there are several differentendogenous retroviral sequences present within the human and felinegenome and that we have amplified only a single one but a different onein each case of human PAC/BAC and feline BAC. To determine which ofthese possibilities is correct, we first need to resolve whether we aredealing with an exogenous or endogenous tetrovirus. The resolution ofthis question will influence subsequent strategies.

In light of data concerning the presence by RT-PCR of gag transcripts incases of human PAC/BAC and feline BAC which are also immunoreactive forJSRV-CA, our findings provide evidence that an exogenous retrovirus isinvolved in either the initiation and/or promotion of human BAC.Moreover, our findings provide evidence that the activation andexpression of an endogenous retrovirus and the gene products of thisretrovirus, e.g., gag in human PAC/BAC is an important step in thepathway of tumorigenesis analogous to the activation of oncogenes. Inaddition, our findings provide evidence that the mechanism ofoncogenesis in human PAC/BAC reactivates retroviral transcripts asdownstream events of the neoplasia. Without being bound by any specifictheory, these downstream events may have nothing to do withtransformation or progression pet se but may reflect the end point of acommon pathway present in both BAC and sheep jaagsiekte (since in sheepjaagsiekte there is also reactivation of endogenous gag).

Using the feline BAC xenograft and cell line and the disclosure providedherein, one can address each of these possibilities by first determiningwhether we are dealing with an exogenous or an endogenous retrovirus. Anumber of procedural steps can be undertaken to address this. If dealingwith an exogenous virus, one skilled in the art can undertake a firststep of recovering a tetrovirus from feline BAC either by a cloningsequence approach or by purifying retroviral particles. If dealing withan exogenous virus, one skilled in the art can undertake a second stepof characterizing the growth-promoting or transforming abilities of theendogenous retrovirus and its gene products (e.g. gag) and a third stepof Detect trans factors in feline BAC which activate endogenousretroviral transcription. Such procedural steps are discussed in detailbelow.

A. Exogenous v Endogenous Retrovirus

We plan to use the 229 bp gag as a probe to do a Southern blot on JSRVpositive BAC and adjacent JSRV-negative normal lung tissues from bothhumans and the SPARKY tumor. With this approach there will be threepossible scenarios:

In a first scenario, the BAC tumoral tissue is positive and the adjacentlung is negative. This is the best scenario because it supports thepresence of an exogenous retrovirus. If this is the case, one can useour 229 bp gag probe and screen a cDNA library (preferably) or a genomiclibrary made from our JSRV positive BAC and clone the complete viralsequence. Or one can use the viral particle approach enumerated inmethodology #1 below.

In a second scenario, the tumoral tissue is negative and the adjacentlung is negative. This will be disappointing because it means that theviral copy numbers are so low (not in every tumor cell) that they can beonly detected by RT-PCR. Our approach will be to amplify other regionsof the retrovirus and clone it.

In a third scenario, the tumoral tissue is positive and the adjacentlung positive. This means that we are likely dealing with an endogenousretrovirus rather than an exogenous one. This situation also exists injaagsiekte or sheep pulmonic adenomatosis where there is reactivation ofa number of endogenous JSRV retroviral transcripts which may play a rolein tumorigenesis. If this scenario turns out to be the case, studiesproposed in methodology #2 and methodology #3, below will be especiallyrelevant.

Methodology #1: Recovery of a Tetrovirus from Human and Feline BACEither by a Cloning Sequence Approach or by Purifying RetroviralParticles.

A) Cloning Sequence Approach

Just as we conducted RT-PCR with primers of gag, we plan to see whetherwe can detect transcripts of pol and env. We anticipate that pol will bepresent and highly conserved but that env will show some divergence. Ifwe are successful we plan to use a nested primer approach and PCR theentire retroviral transcript. Because we have the advantage of havingseveral hundred grams of fresh frozen tissue available from human casesof BAC and SPARKY-X which are JSRV positive by both immunocytochemistryand RT-PCR, one can attempt to isolate an exogenous retrovirus from thistissue with the following approach.

B) Viral Particle Purification Approach

SPARKY-X and those cases of human BAC that give a positive reaction byimmunohistochemistry and reveal JSRV transcripts (preferably with a ScaIsite) by RT-PCR and which abundant fresh-frozen tissue is available canbe examined further to identify the putative virus particles accordingto protocols that have been used successfully to detect retroviruses insheep and goat respiratory tumours (Herring et al., VeterinaryMicrobiology 1983; 8: 237–249; Sharp et al., J of General Virology 1983;64: 2323–2327; De Las Heras et al., J of General Virology 1991: 72:2533–2535; De Las Heras et al., Veterinary Record 1993; 132: 441;Palmarini et al., J of General Virology 1995: 76: 2731–2737). Briefly,tumours can be homogenized (10% w/v suspension in TNE(10 mM Tris, 100 mMNaCl, 1 in M EDTA)) and clarified by centrifugation at 10,000 g/1 h/4°C. The supernatant can be removed and further centrifuged at 100,000×gthrough a double layer of glycerol (25% and 50% v/v) for 1 h at 4° C.The supernate can be removed and the pellet resuspended as a 200 timesconcentrate in TNE buffer. The pellets can examined for retrovirus bywestern blotting as described (Sharp et al., J of General Virology 1983;64: 2323–2327) using the antiserum to JSRV CA. Any samples that producea positive result, indicated by a band of approximate Mr 25–27000reacting with the antiserum to JSRV CA, can be analyzed further byisopycnic centrifugation on 20% to 55% (w/w) sucrose gradients. Thegradients can be fractionated and each 0.5 ml fraction resuspended in4.5 ml TNE buffer, centrifuged at 100,000×g/1 h/4° C. and the resultantpellet resuspended in 100 μl TNE. These fractions can be examined forretrovirus by western blotting as described above. If a positive resultis obtained only in fractions with densities characteristic ofretroviruses, this will support the notion that an exogenous retrovirusis present in these human tumours and attempts can be made toclone/sequence the genome of this virus from this fraction.

Using methods known in the art one can pursue a similar approach inattempting to identify putative retroviral particles from the feline BACcell line and xenograft. In addition to attempting to isolate viralparticles from a homogenate of both cell pellet and xenograft, one canuse both concentrated conditioned media (concentrated 1000 fold usingCentriprep-10 concentrators (Amicon, Beverly, Mass.) and concentratedpleural effusions from BAC tail vein-inoculated Scid mice. This latterapproach may prove more successful in isolating virus because in thejaagsiekte experiments we found that the virus could be more readilyisolated from bronchial and nasal discharge material than from actualtumoral lung tissue. Even though the feline BAC cell line and xenograftcontain no evidence of retroviruses on ultrastructural studies we arenot discouraged by this fact alone because most cases of both natural aswell as experimentally-induced jaagsiekte do not show viral particleswithin tumor cells. If we find evidence of retrovirus in the xenograftsone can be cautious in interpreting these results because we know thatnude and Scid mouse xenografts can be secondarily infected with murineretroviruses. Whether the retrovirus is the JSRV retrovirus can bedetermined by sequencing.

If an exogenous retrovirus appears to be involved, using methods knownin the art one can collect sera prospectively from patients with PAC/BACand screening their sera by Western blot for antibodies against JSRV-CA.If an exogenous retrovirus appears not to be involved in either theinitiation and/or promotion of human/feline BAC, then activation andexpression of an endogenous retrovirus and the gene products of thisretrovirus, e.g. gag or some other retroviral gene product in humanPAC/BAC may nevertheless be an important step in the pathway oftumorigenesis analogous to the activation of oncogenes. For example, inbreast cancer, the int oncogenes were identified solely by theirassociation with mouse mammary tumor virus (MMTV)-induced mammary tumorsin mice and at least one of these oncogenes, int-2, was found to beactivated in some human breast cancers and yet MMTV does not play a rolein human breast cancer. The proposal, in Methodology #2, will examinethis possibility.

Methodology #2: Study the Growth-Promoting or Transforming Abilities ofthe Endogenous Retrovirus and Its Gene Products, e.g. gag

Using methods known in the art one can screen a series of human PAC/BAClines obtained from ATCC and other sources to identify lines thatexpress JSRV gag so that we can work with these as well as our felineBAC line. This approach can utilize both sense and antisense strategieswith JSRV gag and other retroviral genes. One can use recipient humanand feline BAC lines some of which are JSRV positive and some JSRVnegative. Other non-BAC lines, e.g. squamous cell carcinoma can also beused. One can transfect both out JSRV negative/positive cell lines withboth sense and antisense gag constructs appropriately. One can initiallytransfect with electroporation and select the transfectants by G418resistance. The plasmid construct (pBK-CMV), a eukaryotic expressionvectors containing gag cDNA under the control of a CMV promoter as wellas G418 selection can be used. We have determined that with thisapproach our recipient cell lines exhibit a moderate to hightransfection efficiency of 10⁻⁴ and each successfully transfected cloneaverages between 4–10 plasmid copy number.

Screening for successfully transfected clones can be carried out usingour antibodies against JSRV-CA. In the case of antisense transfection wewould of course be looking at a recipient cell line which was initiallyJSRV positive whose transfected subclone became JSRV negative. In thecase of sense transfection we would be looking for exactly the opposite.RT-PCR could also be used but it is not as good as antibody screening.

The JSRV positive and negative clones (as a result of transfection) canbe compared to the natural JSRV positive and negative clones withrespect to growth parameters (tumor cell doubling time, platingefficiency, saturation density, etc.), tumorigenicity, latency, in vivogrowth rate and metastasis. The retroviral gag and other cDNAs which areisolated can also be cloned into inducible expression vectors (eitherusing a modified metallothionine, pSV2M(2)6, or a lactose operonpromoter, pOP13) as an inducible model to investigate the role of thesecDNAs as upstream inducers of other genes related to tumor progressionof BAC. We would then be able to eventually use subtractionhybridization techniques to isolate the genes that are altereddownstream and possibly identify an intracellular pathway which isaffected by retroviral gene expression.

Using methods known in the art our strategy is fairly straightforward.However, if this approach fails to demonstrate that gag or otherretroviral gene products promotes growth or transformation, a thirdhypothesis is that the mechanism of oncogenesis in human and/or felineBAC reactivates endogenous retroviral transcripts as downstream eventsof the neoplasia. These downstream events have nothing to do withtransformation or progression per se but may reflect the end point of acommon pathway present in both human and feline BAC and sheep jaagsiekte(since in sheep jaagsiekte there is also reactivation of endogenousretroviral sequences). This common pathway may be pivotal for tumorprogression in all three diseases. Therefore the activation ofendogenous retroviral transcription may serve as a marker for a moregeneralized pathway of tumor progression in feline, human, and ovineBAC. Methodology #3 is designed to examine this latter hypothesis.

Methodology #3: Detection of Trans Factors in Human and Feline BAC whichActivate Endogenous Retroviral Transcription

In this approach one can use the JSRV positive feline line (SPARKY) andJSRV positive and negative human cell lines of BAC. One can obtain aclone of the JSRV retrovirus from the Moredun Institute. From this cloneone can construct LTR promoter-CAT constructs. In this approach,transfection studies with these constructs can be conducted to identifyboth enhancer and silencer cis elements present in the retroviral LTR.These cs elements can be used to identify trans-acting factors thatenhance retroviral expression in the JSRV positive cell lines andsuppress JSRV expression in the JSRV negative cell lines. All lines canbe transfected with the LTR-CAT constructs and CAT protein can bemeasured. The transfected cells can be standardized by co-transfectionwith a β-galactosidase expression vector, pCH110. This can allow us todetermine the overall activities of JSRV in these cells. We anticipatethat reporter levels can initially reflect the constitutive levels ofJSRV expression in the respective lines. However as we identifydifferent domains of the LTR promoter in particular, we may be able toidentify silencer elements by comparative reduction in CAT levels. Gelmobility shift analyses can then be done. Again the strength of ourapproach is our use of JSRV positive (where LTR-CAT should be high) andnegative cell lines (where LTR-CAT should be low). In the gel mobilityshift assays nuclear cell extracts from the different lines can becompared to each other and to HeLa cells (control protein) (Stratagene,La Jolla, Calif.). If any transcription factor(s) are being expressed inthe JSRV positive cell lines and not the JSRV negative cell lines, wewould be able to determine this using these experiments.

If specific DNA-binding proteins are detected by the above studies theirprecise sequence affinities can be determined by DNA footprintingexperiments. Any protected sites localized and sequenced by thesemethods can be compared to described consensus sequences known to bepresent in other promoters. Confirmation of sites thus defined can beperformed by the use of synthetic oligonucleotides containing theputative control sequence to titrate off the specific DNA bindingprotein(s). Finally an oligonucleotide affinity column can be used topurify the specific DNA binding protein(s) identified. Using analternate approach for identifying the trans-acting factors, we may beable to use these specific oligonucleotides to screen bacterialexpression libraries separately made from cDNA from JSRV positive BACcell lines compared to JSRV negative cell lines. Any cDNAs isolated fromthe expression library can be sequenced/compared to known sequences. Theability of any cDNA to enhance tumorigenicity, growth, etc can beinvestigated in an approach similar to that of Methodology #2.

With the use of these mentioned approaches to define the as-actingelements in the LTR regulatory regions under carefully controlledconditions, it is expected that spurious localization of responsiveelements would be minimized. We could also use RNase protectiontechniques to confirm that increased CAT protein is due to increased CATRNA.

II. Characterization and Culturing Toxoplasma gondii

Toxoplasma gondii is an intracellular protozoan parasite of worldwidedistribution. Currently, parasites that harbor a complete antigenicprofile, that is necessary for the serological diagnosis of humanToxoplasma infections, are provided by in vivo culture methods only.Thus, in most laboratories the asexual proliferative stage of theparasite, the tachyzoite, is maintained by successive intraperitonealpassages in highly susceptible animals such as mice. For references onthe culture of Toxoplasma gondii, see also Klien et al., ALTEX 199815(5): 37–39; Ashburn et al., J Clin Pathol 2000 53(8): 630-3; Evans etal., Eur. J. Clin. Microbiol. Infect. Dis. 1999 18(2): 879–84; Creuzetet al., Parasitol. Res. 1998 84(1): 25–30; Sahm et al., Parasitol. Res.1997 83(7): 659–65; Fischer et al., Parasitol. Res. 1997 83(7): 637–41;James et al., J Clin Microbiol 1996 34(6): 1572–5; Weiss et al., J.Eukaryot Microbiol. 1995 42(2): 150–7 and Couzinet et al., Exp.Parasitol. 1994 78(4): 341–51.

Improved in vitro methods to provide sufficient amounts of high qualityparasite antigen suitable for methods such as vaccine development andproduction is desirable. The invention provide herein allows for avariety of methodological strategies including the growth ofToxoplasmosis in vitro in SPARKY, the growth of Toxoplasmosis in vivo inSPARKY-X in mice and the growth and purification of toxoplasmosis insufficiently large quantities to develop a vaccine.

A typical embodiment of the invention consists of using SPARKY and/orSPARKY-X to culture Toxoplasmosis in vivo and/or in vitro by exposing aculture of SPARKY to Toxoplasmosis sufficient to infect the cells andthen culturing the cells under conditions that permit the growth ofToxoplasmosis. Typical culture conditions for propagating Toxoplasmosisare described above and skilled artisans can readily adapt such methodsfor use with SPARKY and SPARKY-X.

Illustrative Embodiments of the Invention

The invention disclosed herein has a number of embodiments. A preferredembodiment of the invention is a mammalian bronchioloalveolar xenografthaving the biological characteristics of the xenograft designatedSPARKY-X as described in ATCC patent deposit designation PTA-2920 suchthat the xenograft grows within an immunocompromised host in a lepidicbronchioloalveolar carcinoma growth pattern. In highly preferredembodiments, the xenograft is of the type II pneumocyte lineage. Inrelated embodiments, the xenograft expresses a mRNA transcript thatencodes a protein having an amino acid sequence that has at least a 90%identity to the jaagsiekte retrovirus gag amino acid sequence shown inSEQ ID NO: 19. A specific embodiment of the invention is a feline lungcancer xenograft designated SPARKY-X as described in ATCC patent depositNo. PTA-2920.

Yet another embodiment of the invention is a feline bronchioloalveolarcell line having the biological characteristics of the cell linedesignated SPARKY as described in ATCC patent deposit designationPTA-2919 such that the xenograft exhibits surfactant expression as shownby Northern blot and exhibits lamellar bodies ultrastructurally.Typically the cell line expresses a mRNA transcript that encodes aprotein having at least a 90% identity to the jaagsiekte retrovirus gagamino acid sequence shown in SEQ ID NO: 19. A specific embodiment of theinvention is a feline lung cancer cell line designated SPARKY asdescribed in ATCC patent deposit No. PTA-2919.

Yet another embodiment of the invention is a method of generating aSPARKY-X type xenograft by obtaining a sample of bronchioloalveolarcells from the pleural effusion of a mammal having a bronchioloalveolarcarcinoma, implanting the cells from the sample into animmunocompromised mammalian host, and then identifying the xenograftgrowing in the immunocompromised mammalian host

Another embodiment of the invention is a non-human animal model forbronchioloalveolar carcinoma comprising an immunocompromised host animalinoculated with a mammalian bronchioloalveolar xenograft having thebiological characteristics of the xenograft designated SPARKY-X asdescribed in ATCC patent deposit designation PTA-2920 such that thexenograft grows within an immunocompromised host in a lepidicbronchioloalveolar carcinoma growth pattern. Typically theimmunocompromised host animal is a nude mouse. Preferably the mammalianbronchioloalveolar xenograft is the xenograft designated SPARKY-X asdescribed in ATCC patent deposit No. PTA-2920.

Yet another embodiment of the invention is an assay for assessing theeffect of a treatment for bronchioloalveolar carcinoma by applying thetreatment to an immune deficient mouse bearing a bronchioloalveolarcarcinoma xenograft generated by implanting bronchioloalveolar carcinomatissue or a cell suspension thereof from a mammal in the immunedeficient mouse and then determining the effect of the treatment on thegrowth of the xenograft in said mouse. In such assays the treatmenttypically comprises administering a therapeutic agent to the immunedeficient mouse such as a nude mouse. Preferably the mammalianbronchioloalveolar xenograft is the xenograft designated SPARKY-X asdescribed in ATCC patent deposit No. PTA-2920.

Another embodiment of the invention is a method of identifying a mRNAtranscript whose expression is modulated in bronchioloalveolar carcinomaby providing a mammalian bronchioloalveolar xenograft having thebiological characteristics of the xenograft designated SPARKY-X asdescribed in ATCC patent deposit designation PTA-2920 such that thexenograft grows within an immunocompromised host in a lepidicbronchioloalveolar carcinoma growth pattern, determining the level ofexpression of at least one mRNA transcript in the mammalianbronchioloalveolar xenograft, and then comparing the level expression ofthe mRNA transcript in the mammalian bronchioloalveolar xenograft to thelevel of expression of the mRNA transcript in a mammalianbronchioloalveolar cell isolated from a mammal that does not suffer frombronchioloalveolar carcinoma. Typically the level of expression of themRNA of the bronchioloalveolar carcinoma xenograft is determined byNorthern Blotting or polymerase chain reaction. Preferably the mammalianbronchioloalveolar xenograft is the xenograft designated SPARKY-X asdescribed in ATCC patent deposit No. PTA-2920.

A related embodiment of the invention is a method of identifying a mRNAtranscript whose expression is modulated in bronchioloalveolar carcinomaby determining the level of expression of at least one mRNA transcriptin the feline lung cancer cell line designated SPARKY as described inATCC patent deposit No. PTA-2919, and then comparing the levelexpression of the mRNA transcript in the feline lung cancer cell line tothe level of expression of the mRNA transcript in a felinebronchioloalveolar cell isolated from a feline that does not suffer frombronchioloalveolar carcinoma.

Another preferred embodiment of the invention is a method of simulatingthe progression of bronchioloalveolar carcinoma from primary tumorformation to metastasis in an animal model by generating abronchioloalveolar carcinoma xenograft in an immune deficient mouse byimplanting malignant pleural effusion of a BAC bronchioloalveolarcarcinoma, or a cell suspension thereof from a mammal into an immunedeficient mouse and allowing the xenograft to grow for a time sufficientto permit the detection of bronchioloalveolar carcinoma cells withinand/or external to the implant site in the immune deficient mousethereby simulating the progression of bronchioloalveolar carcinoma fromprimary tumor formation to metastasis in the animal model.

Yet another embodiment of the invention is an assay for assessing theeffect of a treatment for bronchioloalveolar carcinoma by applying thetreatment to an immune deficient mouse bearing a bronchioloalveolarcarcinoma xenograft generated by implanting bronchioloalveolar carcinomatissue or a cell suspension thereof from a mammal in the immunedeficient mouse and then determining the effect of the treatment on thegrowth of the xenograft in said mouse.

Yet another embodiment of the invention is an assay for assessing theeffect of a gene of interest on bronchioloalveolar carcinoma byintroducing the gene to an immune deficient mouse bearing a subcutaneousbronchioloalveolar carcinoma xenograft generated by implantingbronchioloalveolar carcinoma tissue or a cell suspension the immunedeficient mouse, transducing the cells of the xenograft with the gene invivo, evaluating the presence of metastasis in the immune deficientmouse by detecting bronchioloalveolar carcinoma cells in the peripheralblood, bone marrow, lymph nodes or other sites distant from the site ofthe subcutaneous xenograft, wherein the effect of the gene on theprogression of bronchioloalveolar carcinoma is determined by referenceto a control immune deficient mouse bearing a subcutaneous human lungxenograft generated with an untransduced subset of the cells of thexenograft. Yet another embodiment of the invention is an a chimericmouse model of bronchioloalveolar carcinoma, said mouse model having abronchioloalveolar carcinoma xenograft, wherein the xenograft in themouse model exhibits a lepidic or alveolar growth pattern characteristicof BAC.

The present invention is further detailed in the following Examples,which are offered by way of illustration and are not intended to limitthe invention in any manner.

Throughout this application various articles, patents, patentapplications, and other publications etc. are referenced. Thedisclosures of these publications etc. are hereby incorporated byreference herein in their entireties. Certain portions of theseincorporated publications etc. may be disclosed for purposes ofproviding a full, dear and concise written description of the variousaspects of the embodiments of the invention.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110–2209, USA (ATCC):

Material ATCC Dep. No. Deposit Date SPARKY PTA-2919 Jan. 19, 2001SPARKY-X PTA-2920 Jan. 19, 2001

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit.

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

EXAMPLES Example 1 Materials and Methods For Characterizing BAC

Total RNA extracted from the BAC cell line was reverse transcribed usingan oligo dT primer (Superscript™, Life Technologies) and then PCRamplified using primers for the feline p53, Hras, K-ras, and N-ras genesas follows:

p53 (1)

AS: 5′-GGCGCCTATGGTTTCCATTTAG-3′ (SEQ ID NO: 3) DR:5′-CATCCAGTGGCTTCTTCTTTTG-3′ (SEQ ID NO: 4)K-ras (5)

IaS: 5′ GACTGAATATAAACTTGTGG 3′ (SEQ ID NO: 5) IIaAS: 5′CTATAATGGTGAATATCTTC 3′ (SEQ ID NO: 6)H-ras (5)

IaS: 5′-GACGGAATATAAGCTGGT-3′ (SEQ ID NO: 7) IIaAS:5′-CCTGTACTGGTGGATGTCC-3′ (SEQ ID NO: 8)N-ras (6)

IaS: 5′-GACTGAGTACAAACTGGTGG-3′ (SEQ ID NO: 9) IIaAS:5′-CTGTAGAGGTTAATATCCGC-3′ (SEQ ID NO: 10)

Conditions of PCR: PCR reactions were performed in 100 microliterscontaining 1× buffer (20 mM Tris-HCl (H 8.4), 50 mM KCl), 1.5 mM MgC12,200 micromolar dNTPs, 200 nM primers and 5 Units of Taq polymerase(Boehringer Mannheim). Cycling for the p53 gene was 30 cycles of 94° C.(1′), 60° C. (1.5′), and 72° C. (2′). The ras genes were similarlyamplified for 35 cycles using a 50° C. (H-ras and K-ras) or 55° C.(N-ras) annealing temperature. DNA sequence analysis was performed on anABI DNA Sequencer Model 377 using 0.1 micrograms of gel purified DNA astemplate for 3.3 pmoles of sense or anti-sense primer. Amplified p53gene segments were also cloned into a Bluescript plasmid (Stratagene)prior to sequencing studies.

The feline p53 primers amplified a 664 by fragment of coding sequence(codons 97–318, exons 4–9) (Okuda et al.,. Int. J. Cancer 58, 602–607(1994); Okuda et al., J. Vet. Med. Sci. 55(5): 801–805 (1993); Mayr etal., Br. Vet J. 151, 707–713 (1995); Mayr et al., Br. Vet J. 151,325–329(1995)). The H-ras, K-ras, and N-ras primers amplified 285, 289, or 289bps, respectively, of exons I and II which contain the mutational hotspot codons 12, 13, and 61 (Watzinger et al., Mamm. Genome 9(3): 214–219(1998); Watzinger et al., Cancer Res. 54: 3934–3938 (1994)).

Summary: DNA sequence analysis of PCR-derived DNA and plasmid cloned DNAof the 664 by fragment the p53 gene repeatedly identified a G:C to T:Atransversion (Arg to Leu) at codon 167 in the BAC cell line, a felinecodon correlate of one of the mutational hot spots reported in humancancers (Okuda et al.,. Int. J. Cancer 58, 602–607 (1994)). A silentmutation was also seen at codon 155 (C:G to T:A), but sequence of normalfeline RNA revealed the same change from the previously publishedsequence. Sequencing studies identified no missense mutations in theH-ras and K-ras genes. Attempts at amplifying the N-ras gene withprimers flanking the first two exons yielded a PCR product whosesequence matched the previously reported feline N-ras pseudogenesequence (Watzinger et al., Mamm. Genome 9(3): 214–219 (1998)). Effortsto specifically amplify the wild type sequence from total RNA wereunsuccessful using the above primers.

Cytogenetics We prepared metaphase slides of the Feline BAC cell linewith a standard technique (Lee et al., M. J. Barch (ed.), The ACTCytogenetics Laboratory Manual pp. 107–148, New York: Raven Press.1991). Briefly, Colcemid (0.1 ug/ml final concentration) was added whencultures were 70–80% confluent. Cells were harvested at 30 minutes, 1hour, and 2 hours following addition of coloemid. We varied the time incolcemid to optimize degree of condensation of metaphase chromosomes forthe purpose of karyotyping. Cells were trypsinized, centrifuged, and thepellet was suspended in hypotonic (0.075 M) KCl for 15 minutes.Following a second centrifugation, cells were fixed in 3:1 methanol:acetic acid. The cell pellet was diluted in the fixing solution to allowappropriate cell concentration and spreading of chromosomes when droppedon microscope slides. Slides were dried and aged for at least one week.

To confirm feline origin, we performed FISH with total feline genomicDNA as a probe. DNA was extracted from the blood lymphocytes of a“normal” cat was used to create the probe using degenerativeoligonucleotide-primed polymerase chain reaction (DOP-PCR) withdigoxigenin-conjugated dUTP (Telenius et al., Genes, Chromosomes &Cancer, 4: 257–263, 1992; Telenius et al., Genomics, 13: 718–725, 1992).Labeled DNA was mixed with 50% formamide, 2×SSC, and 10% dextransulfate, denatured for 10 minutes at 72° C., and preannealed at 37° C.for 45 minutes. No blocking DNA was used. We then allowed the probe tohybridize for 24 hours at 37° C. to denatured metaphase cells from i)the feline BAC cell line, ii) normal feline lymphocytes (positivecontrol), and iii) normal human lymphocytes (negative control). Theslides were washed at 45° C. in 1) 50% Formammide/2×SSC, 2) 2×SSC, 3)0.1×SSC for 10 minutes each. Slides were then rinsed in room temperaturePBD. Slides were treated with FITC-conjugated anti-digoxigenin antibody.Slides were visualized using a Zeiss Axioskop fluorescent microscope.

Chromosomal abnormalities were characterized using a novel method ofcomputer enhanced fluorescent R-banding, developed in our lab (Christianet al., Cytogenetics and Cell Genetics, 82: 1998). In this procedure,metaphase preparations of feline BAC were banded with 4′,6-diamino-2-phenyl-indole (DAPI), which binds A-T rich regions of DNAand with chromomycin A3, which binds preferentially G-C rich regions. Wethen captured gray scale images of a metaphase nucleus for each stainusing a computerized image analysis system. Following an intensitynormalization procedure to account for varying brightness' of thestains, the DAPI image was divided by the Chromomycin image. Thistechnique is similar to that used in CGH. This process gave ahigh-resolution R-banded pattern unique to each chromosome.

Flow Cytometry. A 70–80% confluent culture of the Feline BAC wastrypsinized centrifuged and rinsed in PBS. The cell pellet was thenfixed in cold 70% ethanol. The cell solution was refrigerated for oneday. The cells were then centrifuged and the pellet was dried briefly.The fixed cells were suspended in propidium iodide. This suspension wasfiltered to remove cell clumps. Studies were performed with an EPICS VFlow Cytometer.

Results: FISH performed with labeled feline genomic DNA confirms felineorigin of the cell line. The modal number is 66 based on counts of 30metaphases. This compares with a diploid number of 38 in normal felinecells. The fluorescent R-banding used to characterize cytogeneticaberrations resulted in high-resolution bands. Most abnormalities arenumerical. There are some structural aberrations including an additionof a dark band on the p-arm of an A2 chromosome, an inversion anddeletion in one copy of a C1 chromosome. Flow cytometry confirmedaneuploidy.

Archiving of Human BAC Cases. Previous studies have reviewed andarchived all pathologically verified primary lung cancer cases diagnosedat UCLA Medical Center from 1955 to 1990 (Barsky et al., Cancer 1994;73: 1163–1170; Barsky et al., Modern Path 1994; 7: 633–640). A total of1527 cases were diagnosed during this time period, 1125 (74%) in men and402 (26%) in women. A total of 187 cases, representing 12.2% of all lungcancer cases, were classified as bronchioloalveolar carcinoma. In men,84 cases (7.5%) were of the bronchioloalveolar type. In women, 103 cases(25.6%) were classified as bronchioloalveolar carcinoma. Both the numberand proportion of cases classified as bronchioloalveolar lung cancerhave increased steadily over each five-year time period. The proportionof bronchioloalveolar carcinomas increased from 5% in 1955–1960 to 11%in 1981–1985. Since that time, the proportion has more than doubled,equaling 23% of all lung cancers in 1986–1990. Bronchioloalveolarcarcinoma is now the most common type of lung cancer diagnosed at UCLA,approximately equal in prevalence to squamous cell carcinoma and otheradenocarcinomas. Much of this increase in bronchioloalveolar carcinomahas occurred in women, as evidenced by the female predominance. Whilethe male/female ratio of all other cell types shows a steady declinefrom approximately 7/1 in 1955–1960 to 3/1 in 1986–1990, the male/femaleratio for bronchioloalveolar carcinoma has wavered around unity,dropping below unity for the most recent time period Data from UCLA thenconfirm the increase in bronchioloalveolar carcinoma observed by others(Ikeda et al., Lung cancer 1991; 7:157–164). The cause of this increasein a lung cancer not strongly associated with cigarette smoking is yetto be determined. The cases of human BAC were classified as tocentricity as solitary, diffuse or multifocal. At least some of thecases of multifocal BAC were on the basis of multiclonality (Barsky etal., Cancer 1994; 73: 1163–1170; Barsky et al., Modern Path 1994; 7:633–640). The cases of BAC were also classified according to histologyas sclerotic, non-mucinous and mucinous. The non-mucinous cases werefurther subclassified as Clara, Type II pneumocyte or mixed with thehelp of additional immunocytochemical and ultrastructural studies. Since1990, approximately 200 additional cases of human BAC have beencollected prospectively and classified. A large majority of these caseshave been stored frozen as well as in paraffin. The frozen material haspreserved the DNA, RNA, and protein of human BAC. Demographic data isavailable for most of these cases. Some of these cases reflect verylarge tumors measuring at least 8 cm in greatest dimension and representa source of human material ripe for investigative study.

Example 2 Production of Recombinant JSRV Capsid Proteins

Standard molecular biology procedures were used as described (SambrookJ, Fritsch E, Maniatis T. Molecular cloning: 2nd ed., NY: Cold SpringHarbor Lab, 1989). Briefly, the insert fragment, containing a part ofthe JSRV gag gene (bases 953 to 3030 of the nucleotide sequence (York etal., J of Virology 1992; 66: 4930–4939)) was excised from plasmidpBluescript-Js382 using EcoRI and subcloned into plasmids pMS1S (Sherp Aet al., J of Immunological Methods 1990; 128: 81–87) and pGEX1λT(Pharmacia). These constructs were expressed in E. coli host strainNM522 as β-galactosidase (βgal-CA, plasmid pMCA) andglutathione-S-transferase (GST-CA, plasmid pGCA) fusion proteinsrespectively. Confirmation that the gag gene was in the correct readingframe was obtained by sequencing across the vector-insert junction, aswell testing clones for production of β-galactosidase and GST fusionproteins of the appropriate size by western blotting (immunoblot)analysis with a goat antiserum to Mason-Pfizer monkey virus major capsidprotein (MPMV-CA) (Sharp et al., J of General Virology 1983; 64:2323–2327). Transformed bacteria were grown and induced with isopropylβ-D-thiogalactopyranoside IPTG) for the expression of recombinantproteins. Bacteria were pelleted (5000×g for 10 min) and resuspended in20 ml TE (10 nM Tris pH7.5, 1 mM EDTA). Phenylmethylsulphonylfluoride (2mM) was added before lysing the cell suspension in a French press at1500 psi (10.35 MPa). The lysate obtained was sonicated and clarified at100,000×g at 4° C. for 10 min. βgal-CA fusion protein was purified byaffinity chromatography using a 4 ml column ofaminobenzyl-1-thio-βgalactopyranoside (ABTG) agarose (Sigma) aspreviously described (Cameron et al., Microbiology 1994; 140:1977–1982). GST-CA was purified by affinity chromatography using a 4 mlcolumn of glutathione sepharose (Pharmacia) as recommended by themanufacturers. The yield of soluble βgal-CA was approximately 9 mg/l ofbacterial culture at about 75% purity as estimated by SDS-PAGE. GST-CAcould not be eluted with free glutathione from the Sepharose beads asrecommended by the manufacturer and was therefore further used coupledto the beads to immunize rabbits.

Example 3 Production of Rabbit Polyclonal Antiserum to JSRV-CA

A specific rabbit antiserum to JSRV-CA was prepared by immunizingrabbits with 500 μg βgal-CA combined with Freund's incomplete adjuvant.After 15 days the rabbit was boosted with 500 μg of GST-CA bound to theglutathione Sepharose beads. A third injection of GST-CA (500 μg) wasgiven after 4 weeks and the rabbit was bled 15 days after the lastinjection. By western blotting the rabbit antiserum recognized the tworecombinant proteins βgal-CA and GST-CA, as well as the native 25 kDa CAin JSRV. There was no reaction with any other protein in sucrosegradient purified JSRV. Further specificity of this serum for JSRV-CAhas been verified extensively in the sheep tumour model, using westernblotting, blocking ELISA and immunohistochemistry (Palmarini et al., Jof General Virology 1995: 76: 2731–2737). To minimize non-specificreactions, the antiserum may be absorbed overnight at 4° C. with alysate of IPTG-induced NM522-pMS1S cells. The serum was then centrifugedat 100,000×g/30 min. to pellet any bacterial debris, aliquoted andstored at −20° C. until used. Immunohistochemistry of lung sections ofsheep affected by jaagsiekte revealed in the lungs immunopositivityconfined to the cytoplasm of the transformed Type II pneumocytesinvolved in pulmonary adenomatosis. The immunopositivity was confined tothe neoplastic cells; adjacent interstitial cells and adjacentuntransformed alveolar cells and bronchiolar cells were completelynegative.

Example 4 Demonstration of JSRV-Related Antigen in Cases of Human BACand the Feline BAC Cell Line

Antibodies made to a recombinant JSRV major capsid protein of this virus(derived from gag) in our initial immunocytochemical studiessurprisingly were able to recognize an immunologically related proteinin a significant number of human PAC/BAC cases and in feline BAC but notin other types of lung cancer nor in normal lung. More surprisingly, inour subsequent studies, RT-PCR performed on these PAC/BAC cases revealedexpression of JSRV gag transcripts which were 90–100% identical to boththe endogenous and exogenous gag transcripts (distinguished by a ScaIsite) expressed in sheep jaagsiekte.

Using these rabbit polyclonal antibodies to JSRV-CA, the recombinantJSRV major capsid protein expressed from JSRV gag, we demonstratedintense cytoplasmic immunoreactivity in 23/40 (58/%) cases ofnon-mucinous BAC, in 17/46 (370%) cases of peripheral adenocarcinoma(PAC) and in 2/15 (130%) mucinous BAC. No cases of squamous cell, smallcell, large cell undifferentiated or non-lung carcinomas were positive(0/100 cases). The percentage of JSRV positivity in BAC/PAC increasedover the past three decades (15%–50%). We observed both diffuse andfocal staining within the transformed Type II pneumocytes/Clara cells.Adjacent lung tissues were non-reactive. The feline BAC tumor, derivedcell line (SPARKY) and xenograft (SPARKY-X) also displayed intense focalcytoplasmic JSRV immunoreactivity.

In order to exclude the possibility that the basis of this JSRVimmunoreactivity in the human and feline BAC was a cross-reactingprotein having nothing to due with the presence of the JSRV major capsidprotein, we carried out RT-PCR on the cases of human BAC and the felineBAC cell line that displayed this JSRV immunoreactivity. Cases of humanBAC which were JSRV negative and cases of squamous cell carcinoma andsmall cell carcinoma which were also JSRV negative were also examined byRT-PCR.

Example 5 JSRV-GAG Transcripts by RT-PCR

The genome of the jaagsiekte sheep retrovirus (JSRV) contains thecanonical gag env, and pol genes. The JSRV genome is 7462 nucleotideslong and contains the canonical gag, pol and env genes, as well as anadditional open reading frame (orf-x) that overlaps pol. Reversetranscriptase polymerase chain reaction (RT-PCR) for a 229-bp regioninternal to the gag gene (position 1598 to 1826) was carried out usingprimers (P1) and (P2).

(P1) sense: 5′-GCTGCTTTRAGACCTTATCGAAA-3′ (SEQ ID NO: 1) (P2) antisense:5′-ATACTGCAGCYCGATGGCCAG-3′ (SEQ ID NO: 2)

First strand cDNA was synthesized from total RNA using either randomhexamers, 0.5 μg oligo(dt), or the antisense primer. 0.5 unitInhibit-ACE™ was added. Synthesis of first strand cDNA was performed in20 μl total volume with 0.5 unit Inhibit-ACE™first strand buffer, 10 mMdithiothreitol, 0.5 mM each of dATP, dCTP, dGTP, dTTP, and 200 unitsMMLV reverse transcriptase (at 42° C. for 1 h. The addition of thereverse transcriptase was omitted in control samples to check for DNAcontamination of the RNA preparations. Sheep lung tissues from diseasejaagsiekte lungs served as positive control. PCR cycles employed were94° C. for 1 minute and 35 cycles of 94° C. for 45 sec, 57° C. for 1 minand 72° C. for 1 min, with a final extension of 72° C. for 2 min in aPerkin-Elmer Gen Amp 2400 thermal cycler. PCR products were sequenced byligating in a TA cloning vector (Invitrogen) and analyzed on an AppliedBiosystems Model 373A automated sequencer.

Surprisingly, the RT-PCR performed on representative cases of human BACwhich were JSRV immunopositive revealed the expression of JSRV gagtranscripts which were 95–100% identical to both the endogenous andexogenous gag transcripts (distinguished by a ScaI site) expressed insheep jaagsiekte. The human cases which were analyzed differed slightlyfrom one another and from sheep jaagsiekte tissues. However when theanalyses were repeated on separate days and from starting from frozentissues, 100% agreement in sequences were obtained from the same tissuesource. Hence PCR contamination could be completely excluded. Themajority of the human cases lacked the presence of the ScaI site; onecase however had the ScaI site intact (FIG. 4A). The feline BAC linealso exhibited expression of JSRV gag transcripts containing the ScaIsite which were 91.7% identical to the sheep JSRV (FIG. 3A; FIG. 4A).

A summary of the information provided in FIG. 4A is provided in theTable below.

COMPARISON COMPARISON TO EXOGENOUS TO ENDOGENOUS Mismatches Homology ScaI Site Mismatches Homology BAC 1: 6 97.4% Absent 10 95.6% BAC 2: 4 98.3%Absent 6 97.4% BAC 3: 6 97.4% Absent 8 96.5% BAC 4: 9 96.1% Absent 1095.6% BAC 5: 0 100.0% Present 11 95.2% F BAC: 19 91.7% Present 24 89.5%

In many of these human cases and in the feline line, none of thedisparities in base divergence resulted in a change in amino acidcomposition (FIG. 4B). However one human BAC case had a valinesubstituted for a glycine at bp position 1625–1627 and a valinesubstituted for aspartic add at bp position 1640–1642; another human BAChad only the valine substituted for aspartic add at bp position1640–1642; still a third human BAC had a valine substituted for alanineat bp position 1598–1600 and a valine substituted for aspartic acid atbp position 1649–1651. The ScaI site changed the amino acid at site1724–1726 from glutamine to glutatnic acid (FIG. 4B).

1. A feline bronchioloalveolar carcinoma designated SPARKY-X and havingATCC patent deposit designation PTA-2920.
 2. A feline bronchioloalveolarcell line designated SPARICY and having ATCC patent deposit designationPTA-2919.
 3. A non-human animal model for bronchioloalveolar carcinomacomprising an immunocornpromnised mouse host inoculated with a felinebronchioloalveolar carcinoma xenograft designated SPARKY-X and havinaATCC patent deposit designation PTA-2920.
 4. The animal model accordingto claim 3, wherein the immunocompromised mouse host is a nude mouse. 5.An assay for assessing the effect of a treatment for bronchioloalveolarcarcinoma, comprising: (a) administering a therapeutic agent to animmune deficient mouse bearing a feline bronchioloalveolar carcinomaxenograft generated by implanting SPARKY-X tissue or a cell suspensionthereof in the immune deficient mouse, wherein said tissue having ATCCpatent deposit No. PTA-2920; and (a) determining the effect of the agenton the growth of the xenograft in said mouse.
 6. The assay according toclaim 5, wherein the immune deficient mouse is a nude mouse.
 7. A methodof identifying a mRNA transcript whose expression is modulated inbronchioloalveolar carcinoma comprising the steps of: (a) providing afeline bronchioloalveolar carcinoma xenograft designated SPARKY-X andhaving ATCC patent deposit designation PTA-2920 such that the xenograftgrows within an immunocompromised mouse host in a lepidicbronchioloalveolar carcinoma growth pattern; (b) determining the levelof expression of at least one mRNA transcript in the felinebronchioloalveolar xenograft; and (c) comparing the level expression ofthe mRNA transcript in the feline bronchioloalveolar xenograft to thelevel of expression of the mRNA transcript in a felinebronchioloalveolar cell isolated from a feline that does not suffer frombronchioloalveolar carcinoma.
 8. The method according to claim 7,wherein the level of expression of the mRNA of the bronchioloalveolarcarcinoma xenograft is determined by Northern Blotting or polymerasechain reaction.
 9. A method of identifying a mRNA transcript whoseexpression is modulated in bronchioloalveolar carcinoma comprising thesteps of: (a) determining the level of expression of at least one mRNAtranscript in the feline lung cancer cell line designated SPARKY asdescribed in ATCC patent deposit No. PTA-2919; and (b) comparing thelevel expression of the mRNA transcript in the feline lung cancer cellline to the level of expression of the mRNA transcript in a felinebronchioloalveolar cell isolated from a feline that does not suffer frombronchioloalveolar carcinoma.